Amperometric biosensor in thick film technology

ABSTRACT

Described is an amperometric biosensor in thick film technology for the detection an/or determination of substances undergoing enzyme catalyzed reactions, especially glucose or lactate, comprising an inert carrier material, at least one transducer layer exhibiting a specific electrical conductivity χ&gt;10 4  Ω- 1 cm- 1 , and at least one bioactive layer with diffusion barrier function containing at least one enzyme specifically reacting with said substance to be measured, said bioactive layer exhibiting a specific electrical conductivity χ&lt;1 Ω- 1 cm- 1  and said bioactive layer further effectively hinders other electroactive substances to reach the transducer layer within the time of measurement.

This application is a national phase application under 35 U.S.C. §371 ofPCT Application No. PCT/EP02/07765 filed 12 Jul. 2002, which claimspriority to Austrian Application No. A 1248/2001 filed 9 Aug. 2001, thecontents of which are incorporated herein by reference in theirentirety.

This present invention relates to biosensors in thick film technologyfor the selective detection and/or determination of substancesundergoing enzyme catalyzed reactions, to processes to manufacture suchbiosensors, to screen printing inks suitable for such processes, and tothe synthesis of such printing inks.

Biosensors are based on the coupling of one or more biochemically activesubstances having high selectivity (e.g. enzymes, antibodies etc.) witha physico-chemical transducer followed by electronic signal processingand/or display. Amperometric sensors are used for applications with lowdetection limits (≧10⁻⁸ mol/l) and linear measurement ranges across upto 6 concentration decades. They are based on heterogeneous electrontransfer reactions between the bioactive substances and the transducer.The electrical communication occurs via so called mediators. A naturallyavailable candidate for the function of mediator is the reaction productbetween the bioactive substance and the substance of interest in theanalyte. In complex analytes containing additional electroactivesubstances presently known biosensors suffer from signal interference ifthese substances are capable of reaching the transducer. One concept tosolve this problem involves the addition of alternate mediators, whichhave a lower overvoltage at the electrode and are thus reacting at lowerpotentials than the interfering substances. Using such alternatemediators permits the use of graphite as transducer. It is common to usegraphite, a bioactive substance, and a solvent with other additives toobtain a screen printable ink. This permits to take advantage of thescreen printing technology (e.g. device structuring, economic massproduction etc.) in producing the bioactive layer of sensors. Usingsmall and mobile molecules as alternate mediators, however, introducesthe problem that they are either water soluble and thus leached from thebioactive layer during the measurement (e.g. hexacyanoferate,hydrochinone), or that they are toxic and thus critical for in vivoapplications (e.g. vinylferrocene). Thus it was proposed to avoid theproblem of leaching by covalently binding the mediator to the bioactivesubstance (U.S. Pat. No. 5,804,047). As far is known, sensors producedby this method have an insufficient small linear measurement range.Alternatively, it has been proposed to bind the mediator covalently tothe transducer (AT 397512 B). In the latter case, however, the bioactivelayer is not produceable by screen printing.

There is another concept in biosensor technology which avoids suchalternate mediators (and the accompanying problems): Additionalmembranes are placed either between the analyte and the bioactive layeror between the bioactive layer and the electrode. The firstconfiguration may form an effective diffusion barrier (i.e. theinterfering substances does not alter significantly the signal withinthe time of measurement) for all interfering substances but thesubstance of interest in the analyte, the latter for all interferingsubstances but the mediator. In order to form such an effectivediffusion barrier layer thicknesses around 100 nm typically realizableby drying liquid films (produced e.g. by dip-coating once) or drops areinsufficient. Thick film technology, on the other hand, permits torealize membranes having layer thicknesses ≧5 μm in a single productionstep. As mentioned above, previously published thick film inkformulations for bioactive and/or biocompatible layers in biosensorsalways contain graphite, which in addition functions as transducer. Suchinks are thus not suitable to realize a diffusion barrier between thebioactive substance and the transducer. Without the addition ofgraphite, however, formulations described up to now are not screenprintable, and thus a layer sufficiently thick to form an effectivediffusion barrier cannot be obtained in a single production step.

It is therefore an object of the present invention to provide improvedthick film technology biosensors. Such improved biosensors shouldpreferably be robust, easily produceable also on an industrial scaleand, this would specifically be preferred, graphite free.

Therefore, the present invention provides an amperometric biosensor inthick film technology for the detection an/or determination ofsubstances undergoing enzyme catalyzed reactions, especially glucose orlactate, comprising

-   -   an inert carrier material,    -   at least one transducer layer exhibiting a specific electrical        conductivity χ>10⁴ Ω⁻¹cm^(−1,) serving as electrode, transducer,        etc., and    -   at least one bioactive layer with diffusion barrier function        containing at least one enzyme specifically reacting with said        substance to be measured, said bioactive layer exhibiting a        specific electrical conductivity χ<1Ω⁻¹cm⁻¹ and said bioactive        layer further effectively hinders other electroactive substances        to reach the transducer layer within the time of measurement.

The invention combines the advantages of biosensors (such as e.g. highspecivity in complex media) with the advantages of thick film technology(such as e.g. high flexibility in sensor design, easy integration intoelectrical circuits, mass producible in standardized quality). Thispermits the economic production of sensors for applications in food,biological, pharmaceutical, and medical analysis. Furthermore, theinvention can be employed for monitoring applications ranging from bodyfunctions, to bioreactors, or to the environment.

For immobilisation of the bioactive substance(s) (such as e.g. enzymes)in the bioactive layer the use of one or more biocompatible polymersand/or inorganic gels permits to avoid components in the printing ink,which are necessary to obtain printability but are detrimental for theformation of an affective diffusion barrier between the analyte and thetransducers against interfering electroactive substances.

The production of graphite free, bioactive layers in thick filmtechnology according to the present invention eliminates the occurrenceof interfering signals caused by other electrode active substancespresent in complex analytes without the need to employ alternatemediators.

The production of all sensor layers including the bioactive layers inthick film technology straightforward permits structuring ofminiaturized multisensor arrays. This increases significantly thereliability of the device by structuring several identical sensors inparallel, as well as the selectivity of the device by logicalcombination of several not identical sensors.

The production of the bioactive layer in thick film technologyeliminates problems of inhomogenity in bioacitvity and thus sensitivity.These occur frequently with sensors produced according to the currentstate of the art (using e.g. dip coating etc.). Due to the low cost of(and thus in the electronics industry well established) thick filmtechnology “use once and dispose”—applications can be realized, which upto now could not be considered due to unfavorable economics.

The biosensor according to the present invention may comprise at leastone bioactive layer with diffusion barrier function consisting of two ormore zones, of which the zone adjacent to the electrically conductinglayer is preferably depleted of bioactive substances. However, it isalso preferred to use bioactive layer(s) with homogeneously distributedenzymes contained therein.

According to a further aspect, the present invention relates to aprocess for producing a biosensor according to the present inventionwhich is characterized in that at least one bioactive layer withdiffusion barrier function is produced by screen printing. The presentinvention therefore also relates to a process for producing a biosensoraccording to the present invention, characterized by the following steps

-   -   providing an inert carrier material,    -   depositing at least one electrically conducting layer on this        inert carrier material and    -   depositing at least one bioactive layer with diffusion barrier        function onto the at least one electrically conducting layer by        screen printing using a transducer-free printing ink containing        an enzyme and a biocompatible polymer.

Preferably, at least one bioactive layer is synthesized from a printingink, which contains at least one bioactive substance, but is free ofcomponents, which act as transducers.

Moreover, the present invention relates to a printing ink containing abioactive substance, especially an enzyme, characterized in that it isscreen printable as well as forming bioactive layers with diffusionbarrier function as defined above.

The printing inks according to the present invention conform:

-   -   (a) with the requirements of the screen printing process        (regarding e.g. rheology, adhesion on the substrate etc.), and        after this step form    -   (b) membranes meeting the requirement of the biosensors of        interest (regarding e.g. bioactivity, selectivity, sensitivity,        homogeneity, reliability etc.).

As defined above, the present invention relates to an amperometricbiosensor with the following preferred set-up: On an inert carrier (e.g.Al₂O₃) a working electrode, counter electrode, and reference electrode,as well as the necessary electrical connections are deposited(preferably using screen printing (as e.g. referred to in Whithe “ThickFilm Technology” in Prudenziati (Ed.) “Thick Film Sensors” (Elsevier,Amsterdam, 1994) pp 3ff.)), dried and fixed (by e.g. sintering). Ontothe working electrode a coating of graphite free bioactive materialhaving ≧5 μm layer thickness is deposited (preferably using screenprinting) and dried. This so formed bioactive layer contains bioactivesubstances (such e.g. one or more enzymes, preferably oxidases.) whichare immobilized without the use of heat and UV-radiation by gelentrapment in at least one biocompatible polymer or inorganic gel(preferably polyHEMA, SiO₂).

Putting this bioactive layer in contact with an analyte, which containsthe substrate of at least one of the enzymes immobilized in the layer(such as e.g. glucose for glucose oxidase), this substrate diffuses intothe layer until it is enzymatically reacted. The reaction product (e.g.H₂O₂) functions as mediator formed in situ and diffuses to the workingelectrode serving as transducer.

For such “in situ” mediators the diffusion rates in the biocompatiblepolymer or inorganic gel selected for the production of the bioactivelayer (preferably polyHEMA, SiO₂) are higher, than the diffusion ratesof other electrode active substances (such as e.g. ascorbic acid), whichare typically present in complex analytes and cause interfering sensorsignals at the potential required to detect the “in situ” mediator.Thus, a (polymer and/or gel) layer having ≧5 μm thickness forms aneffective diffusion barrier for such interfering substances and assuresthat within the time of measurement the sensor signal is specificallycaused by the in “situ” mediator. Producing the bioactive layer in thickfilm technology effects that the sensor signal selectively is due to thebioreaction of interest (e.g. oxidation of glucose by glucose oxidase).

In case the substrate for the enzymatic reaction, too, is subjected tothe above described diffusion barrier effect, it may be favorable to addbioactive substances (e.g. enzymes etc.) only to the zone of thebioactive layer adjacent to the analyte, and to reduce its content inthe zone adjacent to the transducer. This technologically more complexproduction process results in advantageous material cost reduction inthe case expensive bioactive substances are involved.

Using screen printing for the production of the bioactive layer resultsin the advantage, that the structuring of the sensor components can beeffected with significant higher resolution (≦10 μm) than withprocesses, which are based on drying of a liquid film or droplet (suchas e.g. dip-coating). This capability of the screen printing technologyto mass produce cost efficiently sensor arrays consisting of severalminiaturized identical sensors in parallel increases significantly thereliability of the device, because the production related non-functionof one individual sensor does not cause the failure of the entiredevice. The just as well cost efficient mass producible logiccombination to an array of several miniaturized not identical sensorspermits the combined detection/determination of more than one substancein the analyte and thus an increase in selectivity beyond one singlebioreaction. This results in a decisive support for the diagnosis ofmedical and biological phenomena.

Producing the bioactive layer by screen printing allows reproduciblecontrol of the homogeneity of the bioactive substance distributiontherein. This permits to avoid inhomogenities, which derive from dryingof layers made by dip-coating or related processes, and which leads tounfavorable variations in sensitivity in so far known sensors.

The screen printing ink, from which sensors characterized by suchadvantageous bioactive layers are made, consists at least of

-   -   (a) one or more bioactive substance (e.g. enzyme, preferably        oxidase) in buffered environment,    -   (b) one or more biocompatible inorganic or organic substance        (e.g. biocompatible polymer, preferably polyHEMA; inorganic gel        former, preferably one or more silicate, aluminate, their        hydrate, hydroxide, alcoholate etc.) to adjust the rheological        properties required for the screen printing process (e.g.        viscosity, thixotropy etc.), and    -   (c) a solvent or mixture of solvents (e.g. water, alcohols,        glycols, polyglycols etc.) which do not physically or chemically        cause deactivation of the bioactive substance(s).

Such inks according to the present invention are free of componentswhich act as transducer. Since so far all screen printed bioactivelayers contain graphite, they do not form effective diffusion barriersagainst interfering substances from the analyse, and sensors madethereof require the addition of alternate mediators for a selectivesignal. According to the present invention these are no more needed.

The preparation of the above described ink advantageously is done byseparate preparation and consecutive controlled unification of solutionsof the biocompatible polymer, inorganic gel former, and bufferedbioactive substance. To prepare solutions of the polymer as well as theinorganic gel former either a previously “ex situ” synthesizedthickening agens (e.g. polyHEMA) may be used, or the thickening agensmay be synthesized “in situ” from the components (e.g. inorganic gelforming oxides, hydroxides, hydrates or alcoholates of Al, B, Ca, Fe, K,Na, Mg, Si, Ti).

The invention is further described in the following examples and thedrawing figures, yet without being restricted thereto.

FIG. 1 shows the rheological behaviour of a printing ink according tothe present invention;

FIG. 2 shows the correlation of glucose concentrations with current; and

FIG. 3 shows the selectivity of the potentiostatically measured signalof a biosensor according to the present invention

EXAMPLES Example 1 Synthesis of a Glucose Oxidase Printing Ink

A polymer solution consisting of 6.625 mass parts polyHEMA (polymerizedhydroxy ethylene metacrylate, molecular weight ˜45000), 59.581 massparts diethylene glycol (DEG), and 33.794 mass parts water is combinedwith 14.959 mass parts SiO₂ gel former. To this solution 720 U/(g ink)glucose oxidase (GOD, EC 1.1.3.4.), are added, typically dissolved in 50mM potassium phosphate buffer pH 7.0 made by mixing of 50 mM KH₂PO₄ and50 mM K₂HPO₄ with water (the pH of the potassium phosphate buffer isadjusted using 3M NaOH). This ink is directly screen printable orstorable at 4° C.

Example 2 Synthesis of a Lactate Oxidase Printing Ink

To a polymer and inorganic gel solution described in Example 1 380 U/(gink) lactate oxidase (LOD, EC 1.1.3.2.), are added, typically dissolvedin 50 mM potassium phosphate buffer pH 7.0 made by mixing of 50 mMKH₂PO₄ and 50 mM K₂HPO₄ with water (the pH of the potassium phosphatebuffer is adjusted using 3M NaOH).

This ink is directly screen printable or storable at 4° C.

Example 3 Synthesis of a Glucose Oxidase Printing Ink

To a polymer solution described in Example 1 3.506 mass parts SiO₂,0.800 mass parts Al₂O₃, 0.040 mass parts Fe₂O₃/FeO, 0.011 mass partsTiO₂, 0.185 mass parts MgO, 0.080 mass parts CaO, and 0.014 mass partsNa₂O gel former are added. Then a glucose oxidase solution described inExample 1 is added. This ink is directly screen printable or storable at4° C.

Example 4 Synthesis of a Lactate Oxidase Printing Ink

To a polymer solution described in Example 1, a gel former described inExample 3 is added. Then a lactate oxidase solution described in Example2 is added. This ink is directly screen printable or storable at 4° C.

Example 5 Rheological Behaviour of a Screen Printable Glucose OxidasePrinting Ink

A screen printing ink consisting of 66.63 mass parts polyHEMA, 89.326mass parts diethylene glycol, and 4.011 mass parts water, 14.959 massparts SiO₂ gel former, and 720 U/(g ink) glucose oxidase (GOD, EC1.1.3.4.), typically dissolved in 50 mM potassium phosphate buffer pH7.0 made by mixing of 50 mM KH₂PO₄ and 50 mM K₂HPO₄ with water (the pHof the potassium phosphate buffer is adjusted using 3M NaOH), has therheological behaviour shown in FIG. 1 and is therefore suitable forscreen printing.

Example 6 Synthesis of a Bioactive Layer Having Controlled LayerThickness by Screen Printing

A screen printing ink according to e.g. Example 1 is placed on top ofthe screen of a screen printing machine, and evenly spreaded out. Theink is then forced through the screen by a squeegee, is allowed to leveland dry. The thickness of the bioactive layer can be controlled by thedrying parameters (Table 1).

TABLE 1 Variation of the layer thickness with the drying temperaturedrying conditions layer thickness 3 days 4° C.  37 μm 3 days roomtemperature 23 μm 3 days 37° C. 22 μm 3 days 80° C. 19 μm

Example 7 Bioactive Glucose Oxidase Thick Film Layer

A sensor with a bioactive layer produced according to Example 6 from ascreen printing ink described in Example 1, contains GOD with anactivity of 80% of the starting activity. The sensor signal is in the μArange, is stable after 20 seconds, and is linear for glucoseconcentrations between 0 and 20 mM (FIG. 2), covering quantitatively therange of physiological interest.

Example 8 Selectivity of the Bioactive Thick Film Layer With GOD

A biosensor with a thick film layer according to Example 7 gives aspecific signal for glucose. This signal is not affected by interferingsubstances such as e.g. ascorbic acid, urea, glycine or paracetamol.FIG. 3 shows the signal for such an electrode measuredpotentiostatically. Curve A: analyte with 5mM glucose (typicalphysiological concentration in blood) an no interfering substances;curve B: analyte with 5 mM glucose plus 60 μM ascorbic acid (maximumphysiological concentration in blood); curve C: analyte with 5 mMglucose plus 25 mM urea (5-fold of the typical concentration in blood),curve D: analyte with 5 mM glucose plus 2,5 mM paracetamol (15-fold ofthe maximum concentration in blood of an arthrosis patient medicatedwith this drug). Even if the figure is magnified (insert in FIG. 3) allcurves are identical within the experimental accuracy.

The invention claimed is:
 1. A thick film amperometric biosensor capableof detection and/or determination of a substance undergoing anenzyme-catalyzed reaction comprising: an inert carrier material; atleast one transducer layer exhibiting a specific electrical conductivityχ>10⁴Ω⁻¹cm⁻¹, during use of the biosensor; and at least one bioactivelayer adapted to function as a diffusion barrier during use of thebiosensor, the bioactive layer further defined as: comprising at leastone enzyme capable of specifically reacting with the substance to bedetected or determined during use of the biosensor; exhibiting aspecific electrical conductivity of χ<1Ω⁻¹cm⁻¹ during use of thebiosensor; and capable of effectively hindering electroactivesubstances, other than any produced by the substance undergoing theenzyme-catalyzed reaction, from reaching the transducer layer during useof the biosensor.
 2. The biosensor of claim 1, further defined asadapted for the detection and/or determination of glucose or lactateundergoing an enzyme-catalyzed reaction.
 3. The biosensor of claim 1,wherein the at least one bioactive layer comprises two or more zones. 4.The biosensor of claim 3, wherein at least one of the at least two ormore zones is a zone adjacent to the electrically conducting layer thatis depleted of bioactive substances.
 5. A process for producing a thickfilm amperometric biosensor capable of detection and/or determination ofa substance undergoing an enzyme-catalyzed reaction comprising:providing an inert carrier material; depositing at least oneelectrically conducting layer on the inert carrier material; anddepositing at least one bioactive layer capable of functioning as adiffusion barrier during use of the biosensor onto the at least oneelectrically conducting layer by screen printing using a transducer-freeprinting ink comprising an enzyme and a biocompatible polymer, thisdepositing being accomplished without the use of UV-radiation, whereinthe bioactive layer is capable of effectively hindering electroactivesubstances, other than any produced by the substance undergoing theenzyme-catalyzed reaction, from reaching the transducer layer during useof the biosensor.
 6. The process of claim 5, wherein the at least onebioactive layer is produced from a printing ink that comprises at leastone bioactive substance but is free of components that could act astransducers during use of the biosensor.
 7. A printing ink comprising anenzyme, wherein the ink is screen printable without the use ofUV-radiation and capable of, in an amperometric biosensor capable ofdetection and/or determination of a substance undergoing anenzyme-catalyzed reaction, forming at least one bioactive layer, adaptedto: function as a diffusion barrier during use of the biosensor; have aspecific electrical conductivity of χ<1 Ω⁻¹cm⁻¹ during use of thebiosensor; and effectively hinder electroactive substances other thanany produced by the substance undergoing the enzyme-catalyzed reactionfrom reaching a transducer layer during use of the biosensor.
 8. Theprinting ink of claim 7, further defined as free of components thatcould act as transducers during use of the biosensor.
 9. A method ofdetection and/or determination of a substance undergoing anenzyme-catalyzed reaction comprising: obtaining a thick filmamperometric biosensor capable of detection and/or determination of asubstance undergoing an enzyme-catalyzed reaction comprising: (a) aninert carrier material; (b) at least one transducer layer exhibiting aspecific electrical conductivity χ>10⁴ Ω⁻¹cm⁻¹, during use of thebiosensor; and (c) at least one bioactive layer adapted to function as adiffusion barrier during use during use of the biosensor, the bioactivelayer further defined as: (i) comprising at least one enzyme capable ofspecifically reacting with the substance to be detected or determinedduring use of the biosensor; (ii) exhibiting a specific electricalconductivity of χ<1 Ω⁻¹cm⁻¹ during use of the biosensor; and (iii)capable of effectively hindering electroactive substances, other thanany produced by the substance undergoing the enzyme-catalyzed reaction,from reaching the transducer layer during use of the biosensor;obtaining a sample; and using the biosensor to detect and/or determine asubstance in the sample.